Potential Roles of CD34⁺ Fibrocytes Masquerading as Orbital Fibroblasts in Thyroid-Associated Ophthalmopathy

Abstract

Context

Orbital tissues in thyroid-associated ophthalmopathy exhibit particular reactivity and undergo characteristic remodeling. Mechanisms underlying these changes have remained largely unexplained. Studies have characterized orbital connective tissues and derivative fibroblasts to gain insights into local manifestations of a systemic autoimmune syndrome.

Evidence Acquisition

A systematic search of PubMed was undertaken for studies related to thyroid-associated ophthalmopathy (TAO), orbital fibroblasts, and fibrocytes involved in pathogenesis.

Evidence Synthesis

Orbital tissues display marked cellular heterogeneity. Fibroblast subsets, putatively derived from multiple precursors, inhabit the orbit in TAO. Among them are cells displaying the CD34⁺CXC chemokine receptor 4⁺collagen I⁺ phenotype, identifying them as fibrocytes, derived from the monocyte lineage. Their unique presence in the TAO orbit helps explain the tissue reactivity and characteristic remodeling that occurs in the disease. Their unanticipated expression of several proteins traditionally thought to be thyroid gland specific, including the TSH receptor and thyroglobulin, may underlie orbital involvement in Graves disease. Although no currently available information unambiguously establishes that CD34⁺ orbital fibroblasts originate from circulating fibrocytes, inferences from animal models of lung disease suggest that they derive from bone marrow. Further studies are necessary to determine whether fibrocyte abundance and activity in the orbit determine the clinical behavior of TAO.

Conclusion

Evidence supports a role for fibrocytes in the pathogenesis of TAO. Recognition of their presence in the orbit now allows development of therapies specifically targeting these cells that ultimately could allow the restoration of immune tolerance within the orbit and perhaps systemically.

Literature pertaining to the putative involvement of bone marrow–derived fibrocytes in thyroid-associated ophthalmopathy is reviewed.

The orbit in Graves disease (GD) exhibits remarkable reactivity (1). In the most severe cases, thyroid-associated ophthalmopathy (TAO) can distort tissue architecture, cause dysfunction, and impair vision. Much of the functional disruption associated with TAO results from end-stage fibrosis. Typically, the course of TAO can be parsed into two clinically recognizable phases, as described by Rundle and Wilson (2). Initially, it presents as a constellation of signs and symptoms attributable to inflammation and localized edema that can extend beyond the anatomic boundaries of the orbit to the upper face. This active phase usually lasts from 1 to 3 years. It gives way to the chronic/stable phase in which the disease ceases changing and when processes, such as fat expansion, muscle enlargement, and fibrosis, are thought to be irreversible. Underlying this characteristic pattern of tissue remodeling appear to be orbital fibroblasts (OFs), which are cells possessing unusual phenotypic attributes. The nature and derivation of OFs have long been speculated about but their precise lineage has remained uncertain. They are generally considered to be neural crest in origin (3). Besides residential OFs, fibroblasts within the orbit could also result from epithelial–mesenchymal transitions (4) or represent cells infiltrating from other anatomic regions, most notably the bone marrow (5, 6).

OFs derived from orbital tissues manifesting TAO exhibit particular cellular heterogeneity and exaggerated responses to many molecular factors in vitro. They hereafter are referred to as GD-OFs. Their unusual characteristics were initially recognized nearly 50 years ago when Sisson et al. (7) began cultivating OFs in vitro. These investigators determined that the release of glycosaminoglycans and glucose utilization in GD-OFs were enhanced when cocultured with lymphocytes (8). These findings remained largely unembellished for nearly two decades before Bahn et al. (9) proposed that cultured GD-OFs might be exploited as a model for studying TAO. Around this same time, Hiromatsu et al. (10) were cultivating OFs and comparing their susceptibility to antibody-dependent cell-mediated cytotoxicity with that of muscle cells. Weetman et al. (11) conducted immunohistochemical analysis of TAO orbital tissues and concluded that the interstitial cells were the likely immune targets in the disease. A number of studies have been performed subsequently examining the ultrastructure of GD-OFs (12, 13), their morphologies (14), and their biochemical attributes (15). Furthermore, it has become clear that GD-OFs derived from orbital fat diverge from those associated with extraocular muscles (14, 16). Of note, GD-OFs express the functional TSH receptor (TSHR) (17, 18). Levels of this receptor are very low compared with those found on thyroid epithelial cells but can be enhanced with their differentiation into adipocytes (19).

Many Aspects of Tissue Remodeling in TAO Can Be Directly Linked to the Phenotypes of GD-OFs

One of the dominant characteristics of fibroblasts in general and GD-OFs in particular concerns their capacity to generate substantial amounts of glycosaminoglycans (20). A cardinal feature of TAO is the disordered accumulation of these macromolecules in the orbit and upper face (20, 21). This material is largely hyaluronan (HA), the most abundant, nonsulfated glycosaminoglycan and one that lacks a core protein (22). HA associates with cellular receptors, CD44 (23) and the receptor for HA-mediated motility (24, 25), and through these interactions initiates cellular responses. It plays a key role in the orbital tissue remodeling and expansion occurring in TAO (26). This can be attributed to its impact on vascular permeability, water content, and the chemoattractant properties of its fragments (27). Its rheological properties include an enormous capacity for water binding. Chain length is an important determinant of the biological impact that HA exerts within tissues (28). Short-chain HA can elicit responses in a wide range of target cell types (29, 30).

Among the early, pivotal insights into the mechanisms underlying HA generation was the subcellular localization of its synthesis to the plasma membrane (31). Subsequently, three mammalian HA synthase (HAS) isoenzymes were identified and cloned (32, 33). The gene encoding uridine 5′-diphosphate (UDP) glucose dehydrogenase was also cloned (34). This enzyme catalyzes the oxidation of UDP glucose to UDP glucuronic acid upstream from the HAS proteins (34). Gene promoters for these enzymes have been cloned (35, 36). HA is produced in the orbit primarily by GD-OFs (20), cells that express all three HAS isoenzymes. HAS2 is the most abundant and appears to dominate HA synthesis in these cells (37). UDP glucose dehydrogenase is also expressed by GD-OFs and can be induced by IL-1β (34, 36). HA synthesis is enhanced in GD-OFs by several cytokines, including IFN-γ (38), leukoregulin (39), CD40 ligand (CD154) (40), and IL-1β (37). A recent study disclosed that IGF-I could also induce HA synthesis in GD-OFs (41) and could skew generation toward high–molecular mass HA in perimysial GD-OFs (42). In contrast, one report found that the induction by IGF-I of HAS2 mRNA in GD-OFs occurred only in the presence of a MAPK inhibitor (43). Forkhead transcription factors have a potentially important role in regulating HA synthesis in these cells (44).

Fat expansion frequently contributes to tissue remodeling in TAO and in some cases predominates in the development of proptosis (45). Adipogenic potential of GD-OFs appears to underlie, at least in part, expansion of orbital tissue (46, 47). These cells express peroxisome proliferator–activated receptor γ (PPARγ). When this receptor is activated, GD-OFs undergo differentiation into triglyceride-accumulating adipocytes (14, 46, 48). Once differentiated, they express elevated levels of TSHR compared with undifferentiated cells (19). It is currently thought that the combination of HA accumulation and adipogenic activity of GD-OF accounts for expansion of orbital connective tissue in TAO.

GD-OFs as Sources of Proinflammatory Cytokines

GD-OFs generate several cytokines that appear to play roles in TAO (1). Many of these have been detected in affected orbital tissues or were found to be generated in activated GD-OFs in vitro. Cytokines detected in situ in TAO include TNF-α, IL-1α, IL-6, IL-8, IL-10, IL-12, IL-13, and IFN-γ (49, 50). Several of these are more highly expressed in active vs stable disease and include IL-1β, IL-6, IL-8, and IL-10 (50). That study also suggested a predominance of T helper (Th)1 cytokines in active TAO. Both IFN-γ and TNF-α induce B cell activating factor in GD-OFs, an induction that appears to be more robust than in cells cultivated from healthy orbital tissues (51). IL-1β induces both IL-16 and RANTES in GD-OFs and in so doing enhances the release of T cell migration-promoting activity (52). The same actions are true for IGF-I and immunoglobulins from patients with GD (53, 54). Additionally, IL-1β, IL-1α, IL-6, IL-1 receptor antagonist, and TGF-β are inducible in GD-OFs (55–57).

GD-OFs Express Molecular Machinery to Generate Arachidonic Acid Derivatives

The repertoire of proinflammatory genes expressed and highly inducible in GD-OFs includes those encoding biosynthetic enzymes for generating prostanoids and eicosanoids. In the cytokine-activated state, GD-OFs produce substantial levels of prostaglandin E₂ (PGE₂) (58) and 15-hydroxyeicosatetraenoic acid (59). This results from active arachidonic acid generation and from the robust induction of several enzymes. CD40 ligand, leukoregulin, and IL-1β induce high levels of prostaglandin endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase (15, 40), and glutathione-dependent PGE₂ synthase (60). Both specific and nonselective cyclooxygenase inhibitors and glucocorticoids attenuate the cytokine-dependent PGE₂ synthesis in GD-OFs. A recent report suggests that sphingosine-1-phosphate (S1P) pathways are involved in mediating the induction of PGHS-2 in GD-OFs (61). That report suggests that the five S1P receptor subtypes are differentially expressed in tissues from patients with TAO compared with healthy orbital tissues. Levels of S1P₁, S1P₂, and S1P₃ receptors are elevated in TAO whereas those for S1P₄ and S1P₅ are higher in healthy tissues. PGE₂ can drive the T cell skew toward a Th2 phenotype and can enhance immunoglobulin synthesis by B cells (62, 63). When engaged with the Th2 cytokine, IL-4, these cells express 15-lipoxygenase-1 (59).

Understanding the heterogeneity of OFs in TAO

GD-OFs are considerably more diverse with regard to their cellular phenotypes in vitro than are fibroblast cultures from healthy orbits and those from other tissues such as skin. Among the first cell markers used to distinguish fibroblast populations was the surface molecule CD90 [thymocyte antigen 1 (Thy-1)], the natural ligand for which remains uncertain but whose role in lymphocyte and thymocyte behavior appears vast (64, 65). Although the proportion of Thy-1⁺ and Thy-1⁻ cells differs among individual donors, most cultures derived from orbital fat can be bisected on the basis of cells displaying that protein (66, 67). In striking contrast, perimysial fibroblasts cultivated from extraocular muscles uniformly display Thy-1 (14). When Thy-1⁺ and Thy-1⁻ GD-OFs were compared side by side, distinct functional differences emerged. Following separation into pure Thy-1⁺ and Thy-1⁻ subsets, both cell populations generate IL-6 when activated by CD40 ligand or IL-1β (67). In contrast, Thy-1⁺ fibroblasts express considerably higher levels of PGHS-2 and generate more PGE₂. Thy-1⁻ fibroblasts synthesize and release more IL-8 under similar culture conditions. Thy-1⁺ cells can differentiate into myofibroblasts when treated with TGF-β whereas Thy-1⁻ fibroblasts accumulate cytoplasmic triglycerides when exposed to PPARγ agonists such as 15-deoxy-Δ^12,14-PGJ₂ or ciglitazone (68). Thus, GD-OFs comprise discrete populations of cells with distinct markers and specialized capacities for biosynthesis and terminal differentiation. An as yet unresolved question concerns whether the different cell types within the TAO orbit derive from a common progenitor or whether some or all GD-OFs originate outside the orbit.

Fibrocytes Play Important Roles in Wound Repair and Fibrosis

Fibrocytes represent a unique, fibroblast-like cell type first described by Bucala et al. (5). They exhibit a CD45⁺CD34⁺CXC chemokine receptor (CXCR)4⁺collagen I⁺ phenotype, the constellation of markers that allows them to be distinguished from other cells with similar morphologies (69). They are relatively rare among circulating mononuclear cells and emanate from the bone marrow where they derive from progenitor cells of the monocyte lineage (69). Their abundance increases in certain diseases such as systemic sclerosis (70) and GD (71, 72). Fibrocytes traffic to sites of tissue injury as a consequence of chemokine networks such as CXC chemokine ligand 12/CXCR4 (73), CC chemokine ligand (CCL)12/CC chemokine receptor (CCR)2 (74), CCL21/CCR7 (75), and CCL3/CCR5 ligand/receptor cognates (76). Important differences in the particular chemokines used for fibrocyte trafficking appear to exist in human beings when compared with mice. They enter injured tissues where they can produce collagen and other extracellular matrix constituents as well as many physiologically important cytokines (77). Several factors appear to determine whether fibrocytes engage in tissue remodeling or exhibit a particular inflammatory phenotype. They are involved in immune responses and host defense. They express major histocompatibility class II (MHC-II) constitutively, as well as an array of costimulatory molecules (78, 79). They also engage in bidirectional crosstalk with T cells. Fibrocytes can prime T cells and depend on contact with lymphocytes that support their differentiation from CD14⁺ monocytes (6). In mice, fibrocyte differentiation from CD11b⁺CD155⁺Gr1⁺ monocytes is dependent on CD4⁺ T cells (80). In general, cytokines such as INF-γ and IL-12 (Th1) inhibit fibrocyte differentiation from monocytes, whereas Th2 cytokines, such as IL-4 and IL-13, enhance that process (81). They participate in the fibrosis associated with several disease processes, including experimental bleomycin-induced lung fibrosis (76). Much of what we currently understand about fibrocyte biology comes from studies of lung fibrosis in mouse models (82). These models are currently being used to identify strategies for minimizing the morbidity associated with fibrocyte-directed fibrosis (83–85).

Fibrocytes exhibit remarkable developmental plasticity and can differentiate into multiple cell types, depending on the intracellular signaling pathways that become activated. These events, in turn, result from extracellular molecular cues coming from their cellular neighborhood. Fibrocytes undergo adipogenesis when exposed to PPARγ agonists (86). In contrast, TGF-β and endothelin 1 drive differentiation toward the myofibroblast phenotype while inhibiting adipogenesis. It is the myofibroblast phenotype, particularly the expression of smooth muscle actin, which conveys the contractile qualities of wound healing associated with fibrocytes. PPARγ activation disrupts the stress-activated protein kinase/Jun N-terminal kinase signaling initiated by TGF-β. Thus, the molecular niche surrounding fibrocytes in the tissues where they infiltrate critically determines their differentiation pathway.

Fibrocytes have been implicated in several disease processes such as those associated with idiopathic pulmonary fibrosis (87). The abundance of CD45⁻collagen I⁺ fibrocytes was found to be elevated in the blood of patients with stable idiopathic pulmonary fibrosis (87). Levels were further increased, up to 10-fold, above those found in stable disease during acute exacerbations. Other comparators, such as patients with adult respiratory distress syndrome, were found to have normal fibrocyte levels (87). Survival of patients with more abundant fibrocytes was considerably shorter than that of individuals with lower levels, suggesting that relative fibrocyte frequency may have predictive value for clinical outcome. Nephrogenic systemic fibrosis occurs in individuals manifesting dermal hardening and thickening associated with renal insufficiency (88). Typically, the affected dermal tissues are infiltrated with fibrocytes, evidence of mucin deposition and thickened collagen bundles. Systemic sclerosis can also be associated with increased levels of fibrocytes (89).

Identifying Fibrocytes as Discrete, Putative Progenitors of a GD-OF Subset in TAO

Fibrocytes have been implicated in the development of multiple ocular-related diseases (90). Among these, diabetic retinopathy development may be mediated at least in part by fibrocytes (91). In that process, fibrovascular membrane formation may result from their activities. Bone marrow–derived cells contributing to the myofibroblast population in corneal wounds (92) exhibit features suggestive of their derivation from fibrocytes. In GD, they have been identified as orbit-infiltrating cells (71). Circulating CD34⁺ fibrocytes become considerably more abundant in patients with GD than in healthy individuals (Fig. 1). Furthermore, it appeared that patients with severe, active TAO exhibited among the highest levels of circulating fibrocytes. CD34⁺ cells have been identified in situ in deep TAO orbital fat. A subpopulation of GD-OFs cultivated from those tissues display CD34 (Fig. 2) (71). A similar fraction of cells failed to surface display CD34, and thus the population of GD-OFs can be subdivided into two discrete subsets with identical morphologies. Whereas the GD-OF population comprises the mixture of CD34⁺ and CD34⁻ cells (∼50% CD34⁺ OFs), those from healthy orbital tissues are uniformly CD34⁻ fibroblasts. It has yet to be established whether CD34⁺ OFs infiltrate the orbit from the circulation or arise from another source. Among CD34⁺ OFs are those that strongly express TSHR. Levels of TSHR are substantially higher in fibrocytes cultivated from blood than in GD-OFs (Fig. 3) and the receptor is considerably more abundant on CD34⁺ OFs than CD34⁻ OFs (93). It is functional in both fibrocytes and GD-OFs; TSH and thyroid-stimulating immunoglobulins (TSIs) induce expression of several cytokines, such as IL-6. TSH induces TNF-α in fibrocytes, a response that is essentially absent in GD-OFs (94). Additionally, signaling initiated through TSHR also results in an induction of IL-1β, IL-1 receptor antagonist, and IL-12 (57, 95, 96). Subjecting fibrocytes to adipogenic culture conditions further enhances TSHR expression. Fibrocyte signaling pathways downstream from TSHR crosstalk with the CXC chemokine ligand 12/CXCR4 network (97), suggesting the potential for TSH/TSI to influence cell trafficking. Fang et al. (98) have reported potentially important interactions between CCR6⁺ Th17 cells and fibrocytes. Circulating Th17 cells are more abundant in TAO and the IL-17 receptor is expressed at higher levels in TAO-derived fibrocytes (99).

Figure 1.

Increased frequency of CD34⁺ fibrocyte generation from the peripheral blood mononuclear cells of 70 patients with GD compared with 25 healthy control donors. P < 0.001. Reproduced with permission from Douglas RS, Afifiyan NF, Hwang CJ, et al. Increased generation of fibrocytes in thyroid-associated ophthalmopathy. J Clin Endocrinol Metab 2010; 95(1):430-438.

Figure 2.

CD34⁺lymphocyte-specific protein-1 (LSP-1)⁺TSHR⁺ fibrocytes can be identified in the orbital tissue of patients with TAO but are absent in tissues from healthy donors. (A) CD34 expression (arrows, green; fluorescein isothiocyanate) in TAO-derived tissue (inset shows negative control staining). (B) Absent CD34 expression in healthy tissue (inset shows positive staining control). (C) LSP-1 expression in TAO-derived tissue (red, arrows; nuclei counterstained with 4′,6′-diamidino-2-phenylindole; inset shows negative control). (D) Absence of LSP-1 expression in healthy tissue (inset shows negative control). (E) CD31 expression in disease-derived tissue is limited to vascular endothelium (red, arrows). (F) Hematoxylin and eosin–stained consecutive thin sections of the same orbital tissue (original magnification, ×40). (G) Fibrocytes present in orbital tissue from patients with TAO coexpress CD34 and TSHR as demonstrated by confocal microscopy. Thin-sectioned tissue from a donor with TAO was stained with anti-CD34 (green) and anti-TSHR (red) antibodies. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole. The inset contains a negative staining control. Reproduced with permission from Douglas RS, Afifiyan NF, Hwang CJ, et al. Increased generation of fibrocytes in thyroid-associated ophthalmopathy. J Clin Endocrinol Metab 2010; 95(1):430-438.

Figure 3.

(A and B) Fibrocytes cultivated from peripheral blood mononuclear cells express high levels of TSHR, regardless of whether they derive from (A) patients with GD or (B) healthy donors. (C) TSHR levels are comparable with those found on primary human thyroid epithelial cells. (D) Undifferentiated OFs fail to express TSHR. (E) Fibrocytes differentiated into adipocytes accumulate intracellular lipid droplets staining with Oil Red O. (F) TSHR levels on fibrocytes remain elevated after differentiation. ISO, isotype control. Reproduced with permission from Douglas RS, Afifiyan NF, Hwang CJ, et al. Increased generation of fibrocytes in thyroid-associated ophthalmopathy. J Clin Endocrinol Metab 2010; 95(1):430-438.

In addition to TSHR, fibrocytes and GD-OFs express high levels of IGF-I receptor (IGF-IR) (100). Inhibiting this receptor activity with the therapeutic monoclonal antibody teprotumumab reduces the surface display of both TSHR and IGF-IR. Furthermore, teprotumumab and another IGF-IR inhibitory monoclonal antibody, 1H7, attenuate the actions of both TSH and IGF-I (41, 95, 100).

Fibrocytes Promiscuously Express Autoantigens

Fibrocytes exhibit a characteristic repertoire of gene expression that includes proteins identified as autoantigens in endocrine autoimmune diseases. They express islet antigen 2 and islet cell autoantigen 69, two self-antigens associated with type I diabetes mellitus (101). Relevant to GD, fibrocytes express not only TSHR but also thyroglobulin (Tg), thyroperoxidase, and sodium iodide symporter (93, 102), proteins initially thought to be restricted to the thyroid gland (93). Their expression is dependent on the noncanonical transcription factor autoimmune regulator protein (AIRE) (Fig. 4). Knocking down AIRE expression with targeting small interfering RNA (siRNA) depresses thyroid protein levels (102). Moreover, fibrocytes from an individual with autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy/dysplasia type 1 syndrome, who harbored a loss-of-function AIRE mutation, were found to express significantly lower levels of these proteins than cells from an unaffected first-degree relative. Preliminary studies suggest that the expression of islet antigen 2 and islet cell autoantigen 69 in fibrocytes may be independent of AIRE (101).

Figure 4.

Interference with AIRE expression knocks down levels of AIRE, Tg, TSHR, sodium iodide symporter (NIS), and thyroperoxidase (TPO). (A) Fibrocytes from four different donors were transfected either with scrambled (control) siRNA or that directed against AIRE. RNA was harvested and subjected to real-time PCR. Data are expressed as mean ± SD. (B) Fibrocyte cultures were transfected with either scrambled siRNA or AIRE-targeting siRNA, and Tg protein was quantified by labeling with [³⁵S]methionine (40 µCi/mL) and then immunoprecipitated. TSHR and NIS were quantified by flow cytometry. Levels of TPO were assessed by Western blotting. Adapted with permission from Fernando R, Lu Y, Atkins SJ, et al. Expression of thyrotropin receptor, thyroglobulin, sodium-iodide symporter, and thyroperoxidase by fibrocytes depends on AIRE. J Clin Endocrinol Metab 2014; 99(7):E1236-E1244.

In contrast to the relatively high levels of thyroid proteins and AIRE expression in fibrocytes, those proteins are either undetectable in GD-OFs or the levels are extremely low (Fig. 5) (93, 102). When separated into pure CD34⁺ OF and CD34⁻ OF subsets, their levels increase substantially in CD34⁺ OFs (93, 102). Furthermore, the amplitude of cytokine induction by TSH is considerably higher in pure CD34⁺ OFs when compared with the mixed population of GD-OFs (94, 103). Thus, CD34⁻ OFs express an inhibitory factor that reduces the expression of several thyroid-related proteins and AIRE as well as cytokine induction by TSH in CD34⁺ OFs (93, 94, 103).

Figure 5.

(A–D) Higher levels of (A) AIRE, (B) MHC-II, (C) Tg, and (D) TSHR in fibrocytes than OFs from patients with GD (GD-OFs). (Left panels) RNA was reverse transcribed and subjected to real-time PCR. (Right panels) Cells were stained with the labeled antibodies for flow cytometry. mRNA data were normalized to their respective glyceraldehyde-3-phosphate dehydrogenase levels and are expressed as the mean ± SD. Data from flow analysis denote fluorescence intensity compared with isotype controls. ***P < 0.001. iso, isotype control. Reproduced with permission from Fernando R, Diniz Grisolia AB, Lu Y, et al. Slit2 modulates the inflammatory phenotype of orbit-infiltrating fibrocytes in Graves’ disease. J Immunol 2018; 200(12):3942-3949. Copyright 2018 The American Association of Immunologists, Inc.

Is Slit2 an Endogenous Fibrocyte Regulatory Factor Within the Orbit in TAO?

Finding that CD34⁻ OFs exert a strong modulatory influence on their CD34⁺ OF counterparts provoked inquiry into the identity and characteristics of this inhibitory activity. That factor appeared to be soluble and released from cell monolayers (93). It could be conveyed to CD34⁺ OFs and circulating fibrocytes by covering those cells with conditioned medium from CD34⁻ OFs (94). Several candidates were considered, including known soluble coactivational molecules involved in the interplay between fibroblasts and lymphocytes (103–106). Proteins exerting influence on cell migration and development were also considered. The axon guidance glycoprotein Slit2 (107, 108) plays critical roles in normal development of the mammalian central nervous system (109). Slit2 imposes the integrity of the midline and enforces the proper organization of the brain. It belongs to a family of three secreted glycoprotein orthologs (110) that bind to receptors known as Roundabouts (ROBOs) (111, 112). ROBOs are transmembrane receptors distributed widely within the central nervous system and in other tissues as well. Slit2 has been shown to also play roles outside the nervous system, such as regulating the inflammatory response (113). It inhibits monocyte chemotaxis through actions mediated by ROBO1 (114) and modulates immune reactivity. Recent evidence suggests that Slit2 can attenuate the differentiation of monocytes into fibrocytes (115). Those pivotal studies indicated that Slit2 generated by fibroblasts surrounding injured lung tissues helps determine the magnitude of fibrosis. Thus, it may represent an important governor of end-stage tissue remodeling and scar formation.

Given the effects of Slit2 on fibrocyte differentiation, examination of its potential impact on the expression of immune- and thyroid-related genes in these cells was undertaken. Slit2 was found to be expressed by GD-OFs, activity that localized specifically to CD34⁻ OFs (103). The protein is highly inducible by TSH and M22, a monoclonal TSI (116), through both transcriptional and posttranscriptional mechanisms. Medium conditioned by CD34⁻ OFs substantially reduces levels of AIRE, Tg, TSHR, and MHC-II when incubated with fibrocytes. Levels of expression can be restored by specifically adsorbing out Slit2 (Fig. 6) (103). Recombinant human Slit2 mimics the actions of the CD34⁻ OF conditioned medium and does so by downregulating the transcription of genes encoding AIRE and Tg (103). Thus, Slit2 appears to represent at least one factor that is expressed and released by CD34⁻ OFs and that may modulate the proinflammatory phenotype of orbit-infiltrating fibrocytes in TAO (Fig. 7).

Figure 6.

Depleting medium conditioned by GD-OFs of Slit2 restores fibrocyte gene expression. (A) Conditioned media from GD-OFs and CD34⁻ OFs were incubated with uncoated beads or beads coated with anti-Slit2 (100 µg), anti-HSP47 (100 µg), or isotype IgG (100 µg). Fibrocyte monolayers were incubated with these media for 5 d. (B) Knocking down Slit2 with specific siRNA enhances gene expression in GD-OFs. GD-OFs were transfected with either scrambled (control) siRNA (3 µg) or Slit2-specific siRNA (3 µg) and incubated 3 d. RNA was extracted, reversed transcribed, and cDNAs were subjected to real-time PCR for the targets indicated. Values were normalized to their respective glyceraldehyde-3-phosphate dehydrogenase levels and expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Reproduced with permission from Fernando R, Diniz Grisolia AB, Lu Y, et al. Slit2 modulates the inflammatory phenotype of orbit-infiltrating fibrocytes in Graves’ disease. J Immunol 2018; 200(12):3942-3949. Copyright 2018 The American Association of Immunologists, Inc.

Figure 7.

Theoretical model for the modulatory role of Slit2 on intraorbital pathogenesis of TAO. When CD34⁻ OF-derived Slit2 predominates, the inflammatory phenotype of CD34⁺ OFs is downregulated and the patient with GD either fails to manifest TAO or the disease is mild (left panel). When CD34⁺ OFs dominate the fibroblast population and the impact of Slit2 is inadequate, TAO is severe (right panel). Reproduced with permission from Fernando R, Diniz Grisolia AB, Lu Y, et al. Slit2 modulates the inflammatory phenotype of orbit-infiltrating fibrocytes in Graves’ disease. J Immunol 2018; 200(12):3942-3949. Copyright 2018 The American Association of Immunologists, Inc.

Fibrocytes and Their Putative Derivative CD34⁺ OFs as Potential Therapeutic Targets in TAO

Given their potential role in the pathogenesis of TAO, fibrocytes might be targeted as a therapeutic strategy. This approach would in theory aim at interrupting both the inflammatory responses and tissue remodeling. The underlying conceptual premise of targeting fibrocytes hinges on their putative regulation of the quality and duration of the tissue disruption and repair. They express several antigens specific to thyroid autoimmunity (71, 93, 102), efficiently present antigens to T cells, display critical coactivational molecules such as CD80 and CD86 (78, 117), and generate an extensive array of cytokines (97, 118, 119). Thus, they could be considered a “switchboard” for the immune responses occurring within the TAO orbit. Recognizing their generation of extracellular matrix molecules, including glycosaminoglycans, fibronectin, and collagen, strongly suggests that modifying these biosynthetic activities could mitigate several adverse consequences of the disease. It is therefore possible that reducing their abundance or behavior, either in circulation or while inhabiting orbital tissue, could alter the disease course and outcome. The endgame would be to reduce disease morbidity and the need for rehabilitative ocular surgeries.

Several factors have been shown to retard fibrocyte differentiation besides Slit2. These factors could prove useful as treatments for TAO. Among them, serum amyloid p (SAP) has been characterized as a fibrocyte-inhibitory agent (120), the actions of which are mediated through Fcγ receptors that are distinct from those blocking neutrophil adhesion (121). A role for SAP in vivo in modulating tissue reactivity was demonstrated in SAP knockout mice (122). These mice exhibit persistent inflammation and fibrosis following administration of the lung fibrosis-promoting agent, bleomycin, when compared with wild-type controls. Very recently, IL-4 was shown to markedly enhance fibrocyte differentiation, an activity that could be inhibited by the store-operated Ca²⁺ entry channel blocker SKF-96365 (123). SAP could block this enhancement by IL-4, apparently utilizing a mechanism mediated through the Ca²⁺ channel. Inhibitors of C-type lectin dendritic cell–specific intracellular adhesion molecule 3–grabbing nonintegrin might also be effective in attenuating fibrocyte differentiation (124). A recent report from Ko et al. (125) provided evidence that blocking the synthesis of S1P and its receptor in GD-OFs could attenuate collagen, fibronectin, and smooth muscle actin synthesis/expression as well as the induction of several metalloproteases by TGF-β. Although not localizing the effects to the CD34⁺ OF subpopulation, these findings suggest that inhibiting this pathway could be related to the activities in fibrocyte-derived cells. Should this prove to be the case, the actions of this agent could prove cell type specific.

Alternatively, fibrocyte activity could be attenuated by interrupting TSHR/IGF-IR postreceptor signaling. Several inhibitory small molecules targeting TSHR have exhibited activity both in vitro and in small animal models (126–129). Monoclonal antibodies that antagonize TSHR activity have also been described (130, 131, 132). It remains possible that one or more of these or similar agents will emerge as well tolerated and effective in treating TAO. Because these molecules should also attenuate the actions of pathogenic TSIs on the thyroid gland and therefore potentially correct hyperthyroidism, they might represent “all-in-one” treatment options for GD. Fibrocyte activity appears to be susceptible to downregulation by specific inhibition of IGF-IR. We have found that immunoglobulins from patients with GD and IGF-I can induce the expression of RANTES and IL-16 in GD-OFs (53, 54). Those and subsequent studies disclosed overexpression of IGF-IR in GD-OFs and T cells and increased abundance of IGF-IR⁺ B cells in GD (54, 133, 134). Furthermore, Tsui et al. (135) reported that IGF-IR and TSHR form physical and functional protein complexes and that inhibition of IGF-IR activity can block signaling downstream from TSHR. These observations represent an important rationale for the use of teprotumumab in treating active TAO. Teprotumumab is a fully human IgG1 that inhibits IGF-IR (136). It has proven safe and effective in a recently completed placebo-controlled clinical trial in moderate to severe, active TAO (137). Cytokine induction by TSIs and TSH in fibrocytes and GD-OFs can be reduced substantially by teprotumumab in vitro (100).

Conclusions

Fibrocytes and derivative CD34⁺ OFs appear to be actively involved in the pathogenesis of TAO as a consequence of their presence in the circulation and orbit. Their repertoire of expressed genes and cellular responses to pathogenic signals insinuates them in the development of this disease. Within tissues, they interact with residential CD34⁻ OFs that condition their behavior. As dominant effectors of pathogenic processes, they appear to possess potential as therapeutic targets. Many aspects of fibrocyte involvement in TAO remain uncertain. Among the most important is whether their relative abundance when compared with CD34⁻ OFs changes as disease activity diminishes, in more severe disease, or in response to therapy. Another relates to whether differences in the phenotype of fibrocytes from one individual to another might underlie the divergent clinical courses of TAO observed in patients. In any event, recognition of fibrocytes and their derivatives as components of orbital biology in health and disease should prove of great importance as we further clarify the process underlying TAO and strive to develop targeted therapies.

Glossary

Abbreviations:

AIRE

autoimmune regulator protein

CCL

CC chemokine ligand

CCR

CC chemokine receptor

CXCR

CXC chemokine receptor

GD

Graves disease

GD-OF

orbital fibroblast from an orbit manifesting TAO

HA

hyaluronan

HAS

hyaluronan synthase

IGF-IR

IGF-I receptor

MHC-II

major histocompatibility class II

OF

orbital fibroblast

PGE₂

prostaglandin E₂

PGHS-2

prostaglandin endoperoxide H synthase-2

PPARγ

peroxisome proliferator–activated receptor γ

ROBO

Roundabout

SAP

serum amyloid P

siRNA

small interfering RNA

S1P

sphingosine-1-phosphate

TAO

thyroid-associated ophthalmopathy

Tg

thyroglobulin

Th

T helper

Thy-1

thymocyte antigen-1

TSHR

thyrotropin receptor

TSI

thyroid-stimulating immunoglobulin

UDP

uridine 5′-diphosphate